Cell

0 Comments

Cell. enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (for schematic details of a simplified two condition comparison (the SILAC medium channel is omitted for clarity). We typically pooled 1/10 of the volume of the pan-specific antibody eluate with the eluates from the modification-specific antibodies for normalization purposes, because the yield of the pan-specific IP was generally much higher. TC-H 106 The pooled eluate was adjusted to contain 1 NuPAGE loading buffer and separated on a 4C12% NuPAGE gradient gel (Invitrogen). Open in a separate window Fig. 1. Antibody evaluation experimental paradigm and method of quantification. 385 to 415, 2) a data-independent MS/MS scan on precursor 394.7, with an isolation width 2.5 and normalized CE 30, scanning from 105 to 815 (designed to catch H3K4me2 in the light SILAC state), 3) a data-independent MS/MS scan on precursor 401.7, with isolation with 2.5 and normalized CE 30, scanning from 110 to 815 (designed to catch H3K4me3 in the light SILAC state), and 4) a full MS scan from 290 to 1700. During the second segment (minutes 20C90), we performed an AIMS experiment (10) where we placed a list of selected precursors on an inclusion list (see supplemental Table 1 for AIMS list). The targets on this list were directed at the SILAC light states of the modified peptides. We acquired a selected ion monitoring MS from 350 to 800 and selected, in intensity order, the top five precursors found in the selected ion monitoring MS scan from the AIMS list for data-dependent MS/MS scans (normalized CE of 30, isolation width of 3.0 values of the peptides can be used to compute the relative enrichment ratio of the targeted histone mark between the mark-specific and pan-specific antibodies. We show in Fig. 1how multiple possible modification states at H3K4 may be monitored in a single LC-MS experiment by generating XICs from their associated precursor ions. We additionally monitor an H3 peptide from residues 41C49 to serve as a proper normalization control. Two representations of the data are shown in the of Fig. 1shows a box that is divided into segments with areas proportional to the amount of ion current from each of the H3K4 modification states from the pan-H3 enrichment (peak area to the peak area for each modification state, normalized by the H3 41C49 peptide ratio. The log2 of this ratio is printed within each box, and the intended enrichment target of the antibody has a of Fig. 1in each heat map. TC-H 106 Where necessary, we collapsed several modification combinations into a single value to represent enrichment of the desired mark (H3K27shows that CST9733 is insensitive to the amino acid at position 31. However, both of the other antibodies actively bias against the form containing Ser-31 regardless of the state. In this sense, MP07-449 could be said to be H3.1/2-selective to a certain degree, whereas CST9733 would give a researcher an unbiased assessment of the state of H3K27me3 regardless of histone variant. H3K9-targeting Antibodies Thus far we have evaluated one H3K9me3-targeting antibody, CST9754 TC-H 106 (Fig. 2H3K4me2, H3K4me3) clustered together and that data from multiple antibodies targeting repressive marks (H3K9me3, H3K27me3) also clustered together. The data from the pan-specific H3 antibody (when compared with itself) fell on the boundary between TC-H 106 these two groups. Open in a separate window Fig. 4. Hierarchical clustering of antibody enrichment histone modification data. corresponds to a particular antibody enrichment strategy and is labeled with the intended target of the antibody and its catalogue number. Each corresponds to a different histone modification, where means unmodified. Each is color-coded by an intensity-weighted average of all modification combinations that bear the mark stated in the column. Intensity of the pan-specific background was used to weight each combination. The depicts a core set of modification enrichments that drive clustering of antibodies together that target canonical activating marks. The depicts a core set of modification enrichments that drive clustering of antibodies together that target canonical repressive marks. (19) noticed that overall levels of both K4 and K79 methylation increased as the acetylation state of H3 increased. Analogously, H3K27me3 and H3K9me3 antibodies co-enriched the second repressive mark along with the Rabbit Polyclonal to OR51E1 targeted one, in Fig. 4in Fig. 4examination of H3K27me3 ChIP tracks obtained using the MP07-449 antibody shows diffuse H3K27me3 regions with highly variability in.